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The inner opercular epithelium of the killifish, Fundulus heteroclitus, may be separated as a single epithelial sheet and mounted in an Ussing-style chamber with the short-circuit current (Isc) approximating chloride secretion. Steady state opercular Isc values in seawater-adapted killifish were higher in tissues removed from fish in the afternoon than those observed in the morning. Opercular conductance was also higher in the afternoon than the morning. The opercular steady state Isc from killifish on a 13:11 light:dark cycle exhibited a diel variation with the peak in the middle of the light cycle and a nadir near the end of the dark phase. Temperature and salinity remained constant during the experimental period. Daily variation cued by the photoperiod should be considered when using this preparation as an experimental model for ion transport in the gill of a seawater-adapted fish.

A procedure was developed for studying vitellogenin (VTG) incorporation by vitellogenic oocytes of Fundulus heteroclitus in vitro. Since homologous VTG can be obtained from this animal only with great difficulty, the use of [32P]VTG from Xenopus laevis was explored as an alternative. Vitellogenic as well as maturational-stage oocytes were found to sequester X. laevis [32P]VTG from the medium, and incorporation was found to be linear with time for at least up to 12 hr. Once incorporated into the oocyte, [32P]VTG did not appear to undergo turnover. The effect of different [32P]VTG concentrations on incorporation indicated that the uptake mechanism was saturable. Unlabeled F. heteroclitus VTG and X. laevis VTG were also found to compete effectively with X. laevis [32P]VTG, whereas bovine serum albumin did not. These results represent the first documentation of a successful culture system for receptor-mediated VTG incorporation by teleost oocytes.

Treatment of the killifish, Fundulus heteroclitus, with ovine thyrotropin (oTSH) produced elevations in serum thyroxin (T4) that varied seasonally with a strong inverse correlation to environmental photoperiod (P less than 0.001). At 20 degrees C, oTSH (0.2 IU) increased serum T4 to levels ranging from 1.5 micrograms/100 ml in midsummer to 7.5 micrograms/100 ml in midwinter despite relatively stable resting levels throughout the two years of study. Similar rates of clearance of T4 in summer and winter suggest that an alteration in thyroid response to oTSH accounts for this change. The serum T4 response to oTSH is both photoperiod- and temperature-dependent. In laboratory studies, fish exposed to a short artificial photoperiod (LD 8:16) for one month responded to oTSH with T4 levels about twice as high as those in fish exposed to long days (LD 14:10). On the other hand the T4 response to oTSH was sharply reduced at 5 degrees C from that seen at 20 degrees C. This suggests that, in nature, seasonal changes in photoperiod and temperature have opposite effects on thyroidal responsiveness to TSH. Overall, the annual variation in the T4 response to oTSH appears to be driven by photoperiod, inasmuch as the alterations preceded major temperature changes in the wild and can be shown to occur at constant temperature in captivity. Photoperiodic induction of changes in thyroid sensitivity may aid in the maintenance of basal T4 levels under changing thermal conditions.

The present study investigates the juvenile and adult reproductive performance of Japanese killifish, Oryzias latipes, which were successfully sex-reversed by feeding them male or female sex hormones during the fry stage. Sexual maturation of these laboratory grown fish of three known genotypes (XX, SY, YY) occurred earliest in untreated genotypes, next in genotypes treated with male hormone (methyl testosterone) and female hormone (estrone), respectively. The delay in sexual maturation caused by early exogenous, sex-hormone treatment may represent a disturbance in the delicate neural-gonadal axis. However, the degree of response was also strongly influenced by the animal's sex chromosomal genotype. XX fish, normally phenotypic females, were affected more by the male hormone than by the female hormone. XY and YY fish, normally phenotypic males, were delayed more by the female hormone than by the male hormone. This specific sex hormone-sex genotype interaction also influenced adult reproductive performance (sperm or egg production). Differences in the metabolism of male and female sex hormones by the XX, XY, and YY fish are probably responsible for these interesting findings.

The present work deals with determination of the threshold of nerve fibers per unit of amputation surface necessary for regeneration of the pectoral fins of a teleost, Fundulus. Partial denervation of the amputated pectoral fins, i.e., resection of one or two of the three nerves of the brachial (=pectoral) plexus revealed that the presence of a single one allows the amputated fin to regenerate. From these data and others obtained previously, it is concluded that the nervous requirements for a teleost fin to regenerate are similar or slightly lower than those for tetrapods, for example in the newt, which are capable of appendage regeneration.

Cell volume regulation has been observed in vitro in many tissues. In the killifish it was studied in vivo during transfer from SW to FW and from FW to SW. It was found that the cell water content in acclimated fish varies inversely with the osmolality of the medium. During transfer from SW to FW the plasma osmolality decreased. Cell swelling was prevented through net loss of solutes from the cells. The solutes lost were not potassium or sodium and are presumed to be organic solutes. Dund no significant increase in cell solute content could be found during eight hours. During prolonged acclimation cell water content returns to a higher value.

A scanning and transmission electron microscope study has been made of the cortical alveoli of the egg of Fundulus heteroclitus. The study includes both unactivated eggs and fertilized eggs fixed at intervals of 1 second to 10 minutes after insemination. The alveoli appear to vary considerably in size, in contents, and in morphological aspects of their breakdown. As it undergoes dehiscence, each vesicle may form one or several openings in the egg surface; dense granules and particulate, fibrous, or membranous material, apparently in any combination, are liberated to the nascent perivitelline space. It appears that much of the excess membrane externalized during the reaction is strung out in threads and probably lost to the perivitelline space. The evidence does not suggest that the excess membrane either "dissolves" or is retrieved by the egg cytoplasm. That part of the cortical vesicle membrane which remains continuous with the oolemma gradually becomes microvillous and loses it morphological identity. Granules and particulate matter, presumably liberated from the cortical alveoli, are seen adhering to the inner surface of chorions removed from activated eggs. The micropyle appears to be sealed with similar material. Supernumerary sperm are observed inside the chorion in some instances. The cortical reaction appears to play secondary role in the prevention of polyspermy and to be somehow related to the subsequent formation of a normal embryonic blastodisc.

The effects of ion substitutions on the Cl- secretion rate and tissue conductance of isolated short-circuited opercular epithelia from sea-water-adapted Fundulus heteroclitus were investigated. Serosal Na+ substitution had the same effect on the Cl- secretion rate that serosal Cl- substitution had on the active component of the Cl- efflux. This similarity indicated a 1:1 Na-Cl requirement for active Cl- secretion across this epithelium, which supports the proposal of a coupled NaCl uptake mechanism at the serosal membrane of Cl- secretory epithelia. Mucosal Na+ and Cl- substitutions appeared to inhibit completely the active Cl- secretory flux. The reductions in the tissue conductance with mucosal ion substitutions suggested that this effect can be attributed to a blocking of the apical membrane Cl- conductance. These mucosal ion effects suggested a possible direct regulatory influence of the external salinity on the Cl- secretion rate and tissue conductance, which provide alternative explanations for observations with the teleost gill epithelium.

Opercular epithelial cells of Fundus heteroclitus were investigated using conventional microelectrodes. The area of interest was the cells lining the inside of the opercular epithelium closest to the gill arches, an area with a high density of chloride cells. Only one cell type could be discerned from the values of 60 opercular cells measured with the opercular epithelium in open circuit conditions. A mean apical voltage of -18.0 +/- 0.6 mV was observed with intracellular values ranging from -10 to -30 mV. The predicted intracellular chloride content was 59 mM/liter. Apical fractional resistance (faR) was 0.78 +/- 0.02. The intracellular potential measurements were typically difficult to maintain for extended periods (longer than 3 min). The opercular cells depolarize with serosal isoproterenol treatment (10(-6) M) corresponding to the increase in opercular transepithelial potential. The opercular cell apical fR decreased with isoproterenol treatment. These data indicate the observed opercular cells were involved in opercular chloride transport.

Several physiological parameters associated with oocyte growth (vitellogenesis) in the killifish, Fundulus heteroclitus, are defined. Hepatocytes from females and estrogen-treated males are highly synthetic cells, whereas those from males are relatively quiescent. A 2000,000-molecular weight phosphoprotein (vitellogenin) has been identified by sodium dodecyl sulfate (SDS)-gel electrophoresis in the plasma of females and estrogen-treated males and isolated chromatographically using a potassium phosphate gradient system on DEAE-cellulose. To inhibit proteolysis, the nontoxic inhibitor, aprotinin, was injected into fish prior to bleeding. The structure of vitellogenic follicles and the process of oocyte growth are described. Once follicles reach a diameter of 0.5 mm, their oocytes incorporate exogenous materials by micropinocytosis and sequestered material is immediately translocated to yolk spheres in the oocyte cortex. This process has been followed by using the tracer horseradish peroxidase. Exogenous proteins appear to leave the perifollicular capillaries via an intercellular route and pass through intercellular channels within the follicular epithelium and patent pore canals of the vitelline envelope before reaching the oocyte surface. [32P]Vitellogenin isolated from F. heteroclitus, [3H]vitellogenin isolated from X. laevis, and the dye trypan blue have been used to determine which sized follicles are vitellogenic (i.e., incorporate exogenous materials into growing oocytes). Morphological and autoradiographic studies are presented to confirm that sequestered macromolecules are incorporated into yolk spheres in the growing oocyte. Follicles appear to grow from 0.5 to 1.3 mm in diameter due to vitellogenesis and subsequently undergo an additional increase in size, which is primarily due to hydration, during oocyte maturation (Wallace and Selman, '78).

Injections of bovine parathyroid hormone (PTH 1-34) and homogenates of corpuscles of Stannius produce hypocalcemia in male killifish and tilapia adapted to calcium-deficient seawater or fresh water, respectively. In fish from water with normal calcium concentrations no effects are noticeable. These results suggest similarity in bioactivity between PTH, the hypercalcemic hormone of terrestrial vertebrates, and the hypocalcemic factor of the corpuscules of Stannius in teleost fish.